ABSTRACT
Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.
ABSTRACT
Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here, we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus T-box binding element (TBE) site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We also demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression , T-Box Domain Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that has been linked to high-fructose corn syrup consumption with induction of hepatic de novo lipogenesis (DNL) as the suggested central mechanism. Feeding diets very high in fructose (> 60%) rapidly induce several features of NAFLD in rodents, but similar diets have not yet been applied in larger animals, such as pigs. With the aim to develop a large animal NAFLD model, we analysed the effects of feeding a high-fructose (HF, 60% w/w) diet for four weeks to castrated male Danish Landrace-York-Duroc pigs. HF feeding upregulated expression of hepatic DNL proteins, but levels were low compared with adipose tissue. No steatosis or hepatocellular ballooning was seen on histopathological examination, and plasma levels of transaminases were similar between groups. Inflammatory infiltrates and the amount of connective tissue was slightly elevated in liver sections from fructose-fed pigs, which was corroborated by up-regulation of macrophage marker expression in liver homogenates. Supported by RNA-profiling, quantitative protein analysis, histopathological examination, and biochemistry, our data suggest that pigs, contrary to rodents and humans, are protected against fructose-induced steatosis by relying on adipose tissue rather than liver for DNL.
Subject(s)
Adipose Tissue/metabolism , Dietary Carbohydrates/adverse effects , Fructose/adverse effects , Lipogenesis , Non-alcoholic Fatty Liver Disease/etiology , Animals , Dietary Carbohydrates/administration & dosage , Disease Models, Animal , Fructose/administration & dosage , Humans , Liver/metabolism , Liver/pathology , Male , Non-alcoholic Fatty Liver Disease/diagnosis , Non-alcoholic Fatty Liver Disease/pathology , Species Specificity , Sus scrofaABSTRACT
Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.
Subject(s)
Antigens, CD/physiology , Antigens, Differentiation, Myelomonocytic/physiology , Atherosclerosis/pathology , Cytokine TWEAK/metabolism , Foam Cells/pathology , Macrophages/pathology , Plaque, Atherosclerotic/pathology , Receptors, Cell Surface/physiology , Animals , Atherosclerosis/etiology , Atherosclerosis/metabolism , Cytokines/metabolism , Female , Foam Cells/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Knockout, ApoE , Plaque, Atherosclerotic/etiology , Plaque, Atherosclerotic/metabolismABSTRACT
The scavenger receptor CD163 is highly expressed in macrophages in sites of chronic inflammation where it has a not yet defined role. Here we have investigated development of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in CD163-deficient C57BL/6 mice. Compared to wild-type mice, the CIA in CD163-deficient mice had a several-fold higher arthritis score with early onset, prolonged disease and strongly enhanced progression. Further, the serum anti-collagen antibody isotypes as well as the cytokine profiles and T cell markers in the inflamed joints revealed that CD163-deficient mice after 52 days had a predominant Th2 response in opposition to a predominant Th1 response in CD163+/+ mice. Less difference in disease severity between the CD163+/+ and CD163-/- mice was seen in the CAIA model that to a large extent induces arthritis independently of T-cell response and endogenous Th1/Th2 balance. In conclusion, the present set of data points on a novel strong anti-inflammatory role of CD163.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Receptors, Cell Surface/deficiency , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Arthritis, Experimental/blood , Arthritis, Experimental/diagnosis , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/pathology , Collagen Type II/immunology , Cytokines/blood , Cytokines/immunology , Cytokines/metabolism , Disease Progression , Humans , Joints/immunology , Joints/pathology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Knockout , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Severity of Illness Index , Synovial Fluid/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th1-Th2 Balance , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
mid and H15 encode Tbx20 transcription factors that specify ventral pattern in the Drosophila leg. We find that there are at least two pathways for mid and H15 specification of ventral fate. In the first pathway, mid and H15 negatively regulate Dpp, the dorsal signal in leg development. mid and H15 block the dorsalizing effects of Dpp signaling in the ventral leg. In loss- and gain-of-function experiments in imaginal discs, we show that mid and H15 block the accumulation of phospho-Mad, the activated form of the Drosophila pSmad1/5 homolog. In a second pathway, we find mid and H15 must also directly promote ventral fate because simultaneously blocking Dpp signaling in mid H15 mutants does not rescue the ventral to dorsal transformation in most ventral leg structures. We show that mid and H15 act as transcriptional repressors in ventral leg development. The two genes repress the Dpp target gene Dad, the laterally expressed gene Upd, and the mid VLE enhancer. This repression depends on the eh1 domain, a binding site for the Groucho co-repressor, and is likely direct because Mid localizes to target gene enhancers in PCR-ChIP assays. A mid allele mutant for the repressing domain (eh1), mideh1, was found to be compromised in gain-of-function assays and in rescue of mid H15 loss-of-function. We propose that mid and H15 specify ventral fate through inhibition of Dpp signaling and through coordinating the repression of genes in the ventral leg.
Subject(s)
Body Patterning/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Extremities/growth & development , Signal Transduction/genetics , T-Box Domain Proteins/genetics , Animals , Animals, Genetically Modified , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Imaginal Discs/growth & development , Imaginal Discs/metabolism , Mutation , T-Box Domain Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Hemoglobin (Hb) toxicity precipitates secondary brain damage following intracerebral hemorrhage (ICH). CD163 is an anti-inflammatory Hb scavenger receptor and CD163-positive macrophages/microglia locally accumulate post-bleed, yet no studies have investigated the role of CD163 after ICH. ICH was induced in wildtype and CD163-/- mice and various anatomical and functional outcomes were assessed. At 3 d, CD163-/- mice have 43.4 ± 5.0% (p = 0.0002) and 34.8 ± 3.4% (p = 0.0003) less hematoma volume and tissue injury, respectively. Whereas, at 10 d, CD163-/- mice have 49.2 ± 15.0% larger lesions (p = 0.0385). An inflection point was identified, where CD163-/- mice perform better on neurobehavioral testing and have less mortality before 4 d, but increased mortality and worse function after 4 d (p = 0.0389). At 3 d, CD163-/- mice have less Hb, iron, and blood-brain barrier dysfunction, increased astrogliosis and neovascularization, and no change in heme oxygenase 1 (HO1) expression. At 10 d, CD163-/- mice have increased iron and VEGF immunoreactivity, but no significant change in HO1 or astrogliosis. These novel findings reveal that CD163 deficiency has distinct temporal influences following ICH, with early beneficial properties but delayed injurious effects. While it is unclear why CD163 deficiency is initially beneficial, the late injurious effects are consistent with the key anti-inflammatory role of CD163 in the recovery phase of tissue damage.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Cerebral Hemorrhage/genetics , Receptors, Cell Surface/genetics , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Blood-Brain Barrier , Brain/pathology , Cerebral Hemorrhage/metabolism , Cerebral Hemorrhage/psychology , Gliosis/etiology , Gliosis/genetics , Hematoma/genetics , Hematoma/pathology , Hemoglobins/metabolism , Iron/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic/genetics , Psychomotor Performance , Receptors, Cell Surface/metabolism , Recovery of FunctionABSTRACT
Increased consumption of high-caloric carbohydrates contributes substantially to endemic non-alcoholic fatty liver disease in humans, covering a histological spectrum from fatty liver to steatohepatitis. Hypercaloric intake and lipogenetic effects of fructose and endotoxin-driven activation of liver macrophages are suggested to be essential to disease progression. In the present study, we show that a low dose of an anti-CD163-IgG-dexamethasone conjugate targeting the hemoglobin scavenger receptor CD163 in Kupffer cells and other M2-type macrophages has a profound effect on liver inflammatory changes in rats on a high-fructose diet. The diet induced severe non-alcoholic steatohepatitis (NASH)-like changes within a few weeks but the antibody-drug conjugate strongly reduced inflammation, hepatocyte ballooning, fibrosis, and glycogen deposition. Non-conjugated dexamethasone or dexamethasone conjugated to a control IgG did not have this effect but instead exacerbated liver lipid accumulation. The low-dose anti-CD163-IgG-dexamethasone conjugate displayed no apparent systemic side effects. In conclusion, macrophage targeting by antibody-directed anti-inflammatory low-dose glucocorticoid therapy seems to be a promising approach for safe treatment of fructose-induced liver inflammation.
ABSTRACT
Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.
Subject(s)
ATP-Binding Cassette Transporters/deficiency , Liver/metabolism , Metabolomics/methods , ATP-Binding Cassette Transporters/genetics , Animals , Cytosol/chemistry , Mice , Multidrug Resistance-Associated Proteins , Mutation , Pseudoxanthoma Elasticum/geneticsABSTRACT
The Drosophila Tbx20 homologs midline and H15 act as selector genes for ventral fate in Drosophila legs. midline and H15 expression defines the ventral domain of the leg and the two genes are necessary and sufficient for the development of ventral fate. Ventral-specific expression of midline and H15 is activated by Wingless (Wg) and repressed by Decapentaplegic (Dpp). Here we identify VLE, a 5â kb enhancer that drives ventral specific expression in the leg disc that is very similar to midline expression. Subdivision of VLE identifies two regions that mediate both activation and repression and third region that only mediates repression. Loss- and gain-of-function genetic mosaic analysis shows that the activating and repressing regions respond to Wg and Dpp signaling respectively. All three repression regions depend on the activity of Mothers-against-decapentaplegic, a Drosophila r-Smad that mediates Dpp signaling, and respond to ectopic expression of the Dpp target genes optomoter-blind and Dorsocross 3. However, only one repression region is responsive to loss of schnurri, a co-repressor required for direct repression by Dpp-signaling. Thus, Dpp signaling restricts midline expression through both direct repression and through the activation of downstream repressors. We also find that midline and H15 expression are both subject to cross-repression and feedback inhibition. Finally, a lineage analysis indicates that ventral midline-expressing cells and dorsal omb-expressing cells do not mix during development. Together this data indicates that the ventral-specific expression of midline results from both transcriptional regulation and from a lack of cell-mixing between dorsal and ventral cells.
ABSTRACT
AIM: The Pringle maneuver is a way to reduce blood loss during liver surgery. However, this may result in ischemia/reperfusion injury in the development of which Kupffer cells play a central role. Corticosteroids are known to have anti-inflammatory effects. Our aim was to investigate whether a conjugate of dexamethasone and antibody against the CD163 macrophage cell surface receptor could reduce ischemia/reperfusion injury in the rat liver. METHODS: Thirty-six male Wistar rats were used for the experiments. Animals were randomly divided into four groups of eight receiving anti-CD163-dexamethasone, high dose dexamethasone, low dose dexamethasone or placebo intravenously 18 h before laparotomy with subsequent 60 min of liver ischemia. After reperfusion for 24 h the animals had their liver removed. Bloods were drawn 30 min and 24 h post ischemia induction. Liver cell apoptosis and necrosis were analyzed by stereological quantification. RESULTS: After 24 h' reperfusion, the fraction of cell in non-necrotic tissues exhibiting apoptotic profiles was significantly lower in the high dose dexamethasone (p = 0.03) and anti-CD163-dex (p = 0.03) groups compared with the low dose dexamethasone and placebo groups. There was no difference in necrotic cell volume between groups. After 30 min of reperfusion, levels of haptoglobin were significantly higher in the anti-CD163-dex and high dose dexamethasone groups. Alanine aminotransferase and alkaline phosphatase were significantly higher in the high dose dexamethasone group compared to controls after 24 h' reperfusion. CONCLUSIONS: We show that pharmacological preconditioning with anti-CD163-dex and high dose dexamethasone reduces the number of apoptotic cells following ischemia/reperfusion injury.
ABSTRACT
The aim of this prospective open-label study was to treat disabling drooling in children with cerebral palsy (CP) with onabotulinumtoxin A (A/Ona, Botox®) into submandibular and parotid glands and find the lowest effective dosage and least invasive method. A/Ona was injected in 14 children, Mean age 9 years, SD 3 years, under ultrasonic guidance in six successive Series, with at least six months between injections. Doses and gland involvement increased from Series A to F (units (U) per submandibular/parotid gland: A, 10/0; B, 15/0; C, 20/0; D, 20/20; E, 30/20; and F, 30/30). The effect was assessed 2, 4, 8, 12, and 20 weeks after A/Ona (drooling problems (VAS), impact (0-7), treatment effect (0-5), unstimulated whole saliva (UWS) flow and composition)) and analyzed by two-way ANOVA. The effect was unchanged-moderate in A to moderate-marked in F. Changes in all parameters were significant in E and F, but with swallowing problems ≤5 weeks in 3 of 28 treatments. F had largest VAS and UWS reduction (64% and 49%). We recommend: Start with dose D A/Ona (both submandibular and parotid glands and a total of 80 U) and increase to E and eventually F (total 120 U) without sufficient response.
Subject(s)
Botulinum Toxins, Type A/therapeutic use , Cerebral Palsy/drug therapy , Sialorrhea/prevention & control , Adolescent , Botulinum Toxins, Type A/administration & dosage , Botulinum Toxins, Type A/adverse effects , Cerebral Palsy/complications , Child , Child, Preschool , Dose-Response Relationship, Drug , Female , Humans , Longitudinal Studies , Male , Parotid Gland/drug effects , Prospective Studies , Sialorrhea/etiology , Submandibular Gland/drug effects , Treatment OutcomeABSTRACT
INTRODUCTION: Surgical resection is the gold standard in treatment of hepatic malignancies, giving the patient the best chance to be cured. The liver has a unique capacity to regenerate. However, an inflammatory response occurs during resection, in part mediated by Kupffer cells, that influences the speed of regeneration. The aim of this study was to investigate the effect of a Kupffer cell targeted anti-inflammatory treatment on liver regeneration in rats. METHODS: Two sets of animals, each including four groups of eight rats, were included. Paired groups from each set received treatment with placebo, low dose dexamethasone, high dose dexamethasone or low dose anti-CD163 dexamethasone. Subsequently, the rats underwent 70% partial hepatectomy. The two sets were evaluated on postoperative day 2 or 5, respectively. Blood was drawn for circulating markers of inflammation and liver cell damage; liver tissue was sampled for analysis of regeneration rate and proliferation index. RESULTS: The high dose dexamethasone group had significantly lower body and liver weight than the placebo and anti-CD163-dex groups. There were no differences in liver regeneration rates between groups. Hepatocyte proliferation was completed faster in the placebo group, although this was not significant. The anti-CD163-dex group showed increased blood levels of albumin and alanine aminotransferase and a diminished inflammatory response in terms of significantly reduced haptoglobin, α2-macroglobulin and Interleukine-6. CONCLUSION: Low dose dexamethasone targeted to Kupffer cells does not affect histological liver cell regeneration after 70% hepatectomy in rats, but reduces the inflammatory response judged by circulating markers of inflammation.
ABSTRACT
PURPOSE: The hemoglobin scavenger receptor CD163 is exclusively expressed on monocytes and tissue macrophages, also at sites of inflammation. We examined whether gallium-68 (Ga-68)-labeled anti-CD163-antibody can detect the receptor in vivo. PROCEDURES: We radiolabeled anti-CD163 antibody with Ga-68 and evaluated stability and binding specificity of the conjugate ([(68)Ga]ED2) in vitro. Furthermore, tracer biodistribution was assessed in vivo in healthy rats and rats with acute collagen-induced arthritis (CIA) by MicroPET and tissue analysis. RESULTS: Radiosynthesis of [(68)Ga]ED2 antibody yielded a tracer with high-specific activity and radiochemical purity. [(68)Ga]ED2 bound specifically to CD163 in vitro. In rats, [(68)Ga]ED2 rapidly accumulated in macrophage-rich tissues. The arthritic paws exhibited a low but significant [(68)Ga]ED2 uptake. Interestingly, the systemic distribution was also changed in the sense that a significantly higher liver uptake and lower spleen uptake of [(68)Ga]ED2 was measured in CIA rats that accordingly showed a corresponding change in level of CD163 expression. CONCLUSIONS: [(68)Ga]ED2 specifically binds CD163 in vitro and in vivo. Biodistribution studies in CIA rats suggest that this novel tool may have applications in studies of inflammatory diseases.
Subject(s)
Antigens, CD/chemistry , Antigens, Differentiation, Myelomonocytic/chemistry , Arthritis/diagnostic imaging , Collagen/chemistry , Gallium Radioisotopes/chemistry , Positron-Emission Tomography , Receptors, Cell Surface/chemistry , Animals , Arthritis/chemically induced , CHO Cells , Cricetinae , Cricetulus , Disease Models, Animal , Female , Gallium/chemistry , Hemoglobins/chemistry , Heterocyclic Compounds/chemistry , Heterocyclic Compounds, 1-Ring , Inflammation , Macrophages/diagnostic imaging , Macrophages/pathology , Rats , Rats, Inbred LewABSTRACT
The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Inflammation/metabolism , Macrophages/metabolism , Receptors, Cell Surface/metabolism , Tumor Necrosis Factor-alpha/metabolism , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM17 Protein , Amino Acid Sequence , Animals , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Binding Sites/genetics , Cells, Cultured , Endotoxemia/genetics , Endotoxemia/metabolism , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , HEK293 Cells , Humans , Immunoblotting , Inflammation/genetics , Lipopolysaccharides/pharmacology , Macrophages/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutation , Proteolysis/drug effects , RNA Interference , Receptors, Cell Surface/genetics , Sequence Homology, Amino Acid , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
OBJECTIVES: Macrophages are important cells in immunity and the main producers of pro-inflammatory cytokines. The main objective was to evaluate if specific delivery of glucocorticoid to the macrophage receptor CD163 is superior to systemic glucocorticoid therapy in dampening the cytokine response to lipopolysaccharide infusion in pigs. DESIGN: Two randomized, placebo-controlled trials. SETTING: University hospital laboratory. SUBJECTS: Female farm-bred pigs (26-31 kg). DESIGN: A humanized antibody that binds to pig and human CD163 was produced, characterized, and conjugated with dexamethasone. In the first study (total n = 12), pigs were randomly assigned to four groups: 1) saline; 2) dexamethasone (1.0 mg/kg); 3) dexamethasone (0.02 mg/kg); and 4) anti-CD163-conjugated dexamethasone (0.02 mg/kg). In the second study (total n = 36), two additional groups were included in addition to the four original groups: 5) anti-CD163-conjugated dexamethasone (0.005 mg/kg); 6) unconjugated anti-CD163. Treatments were given 20 hours prior to infusion of lipopolysaccharide (1 µg × kg × h) for 5 hours. Blood samples were analyzed for cytokines, cortisol, and adrenocorticotropic hormone. RESULTS: In the saline group, lipopolysaccharide increased cytokine and plasma cortisol levels. In both studies, dexamethasone (1 mg/kg) and anti-CD163 dexamethasone (0.02 mg/kg) uniformly attenuated tumor necrosis factor-α peak levels (both p < 0.05) compared with low-dose dexamethasone (0.02 mg/kg). However, dexamethasone 1 mg/kg significantly suppressed plasma cortisol and adrenocorticotropic hormone levels compared with anti-CD163 dexamethasone (0.02 mg/kg; p < 0.05). No significant hemodynamic difference existed between groups. The anti-CD163 dexamethasone drug conjugate exhibited a fast plasma clearance, with a half-life of approximately 5-8 minutes. CONCLUSION: Targeted delivery of dexamethasone to macrophages using a humanized CD163 antibody as carrier exhibits anti-inflammatory effects comparable with 50 times higher concentrations of free dexamethasone and does not inhibit endogenous cortisol production. This antibody-drug complex showing similar affinity and specificity for human CD163 is, therefore, a promising drug candidate in this novel type of anti-inflammatory therapy.
Subject(s)
Antigens, CD/administration & dosage , Antigens, Differentiation, Myelomonocytic/administration & dosage , Dexamethasone/administration & dosage , Drug Carriers/pharmacology , Endotoxemia/drug therapy , Glucocorticoids/administration & dosage , Macrophages/metabolism , Receptors, Cell Surface/administration & dosage , Animals , Antigens, CD/pharmacology , Antigens, Differentiation, Myelomonocytic/pharmacology , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Female , Glucocorticoids/pharmacology , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , Random Allocation , SwineABSTRACT
The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO), biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6), but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Haptoglobins/metabolism , Heme Oxygenase-1/metabolism , Hemoglobins/metabolism , Receptors, Cell Surface/metabolism , Animals , Haptoglobins/chemistry , Heme/metabolism , Humans , Protein BindingABSTRACT
AIM: In humans, plasma haptoglobin (Hp) and the macrophage receptor CD163 promote a fast scavenging of hemoglobin (Hb). In the present study, we have compared the mouse and human CD163-mediated binding and uptake of Hb and HpHb complex in vitro and characterized the CD163-mediated plasma clearance of Hb in CD163 gene knockout mice and controls. RESULTS: Contrary to human Hp, mouse Hp did not promote high-affinity binding to CD163. This difference between mouse and man was evident both by analysis of the binding of purified proteins and by ligand uptake studies in CD163-transfected cells. Plasma clearance studies in mice showed a fast clearance (half-life few minutes) of fluorescently labeled mouse Hb with the highest uptake in the kidney and liver. HPLC analysis of serum showed that the clearance curve exhibited a two-phase decay with a faster clearance of Hb than plasma-formed HpHb. In CD163-deficient mice, the overall clearance of Hb was slightly slower and followed a one-phase decay. INNOVATION AND CONCLUSION: In conclusion, mouse Hp does not promote high-affinity binding of mouse Hb to CD163, and noncomplexed mouse Hb has a higher CD163 affinity than human Hb has. Moreover, CD163-mediated uptake in mice seems to only account for a part of the Hb clearance. The new data further underscore the fact that the Hp system in man seems to have a broader and more sophisticated role. This has major implications in the translation of data on Hb metabolism from mouse to man.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Haptoglobins/metabolism , Hemoglobins/metabolism , Receptors, Cell Surface/genetics , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , CHO Cells , Cricetinae , Cricetulus , Gene Expression , Gene Targeting , Genotype , Humans , Mice , Mice, Knockout , Multiprotein Complexes/metabolism , Protein Binding , Receptors, Cell Surface/metabolism , Surface Plasmon ResonanceABSTRACT
The regulation of the segment polarity gene wingless is essential for the correct patterning of the Drosophila ectoderm. We have previously shown that the asymmetric activation of wingless downstream of Hedghog-signaling depends on the T-box transcription factors, midline and H15. Hedgehog activates wingless anterior to the Hedgehog domain. midline/H15 are responsible in part for repressing wingless in cells posterior to the Hedgehog expressing cells. Here, we show that Midline binds the Groucho co-repressor directly via the engrailed homology-1 domain and requires an intact engrailed-homology-1 domain to repress wingless. In contrast, the regulation of Serrate, a second target of midline repression, is not dependent on the engrailed-homology-1 domain. Furthermore, we identify a midline responsive region of the wingless cis-regulatory region and show that Midline binds to sequences within this region. Mutating these sequences in transgenic reporter constructs results in ectopic reporter expression in the midline-expression domain, consistent with wingless being a direct target of Midline repression.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Repressor Proteins/metabolism , T-Box Domain Proteins/metabolism , Wnt1 Protein/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Body Patterning/genetics , Body Patterning/physiology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , DNA Primers/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Genes, Insect , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Interaction Domains and Motifs , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Homology, Amino Acid , Serrate-Jagged Proteins , Signal Transduction , T-Box Domain Proteins/chemistry , T-Box Domain Proteins/genetics , Wnt1 Protein/chemistry , Wnt1 Protein/geneticsABSTRACT
Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.